Cytochrome P450IA1 and P450IA2 are felt to play an important role in the etiology of cancer, for these proteins in experimental animals and humans have been shown to metabolize PAHs and heterocyclic amines to agents that are toxic and mutagenic. The carcinogenicity of arylamines has been well established in both humans and animals. The metabolic N-oxidation of arylamines is a critical activation step leading to carcinogenesis. One of the primary proteins in humans that maintains this substrate specificity is P450IA2. In most species, P450IA1 is not expressed constitutively, and in humans this protein has not yet been identified. However, P450IA2 is present in considerable abundance in human and animal liver. In addition, the expression and induction of P450IA2 is restricted primarily to the liver. Since P450IA2 is the predominant P450IA protein in humans, experiments are outlined to study the molecular mechanisms that control the expression of the human CYP1A2 gene. The human CYP1A2 gene has been cloned and characterized. A 300 base pair region >2000 bases from the promoter in the 5' flanking region of the gene has been shown to support PAH induction of an reporter gene only in liver derived cell lines. This result indicated that PAH responsive CYP1A2 gene was regulated in a tissue-specific fashion. Employing DNA transfection techniques, two regions of the enhancer sequence have been identified that are necessary for the tissue specific expression by PAH. One region requires the participation of a 3MC-inducible protein while the other requires a liver specific tissue factor. In this application, we will be generating selective base pair deletions through regions of the enhancer sequence. A series of experiments are proposed to identify and characterize the two cis-acting elements. Using techniques such as DNA footprint analysis and methylation interference, the precise location of these two regulatory elements will be determined. In addition, to verify that the trans-acting factors support tissue specific control, gene constructs are also being made and techniques developed to conduct in vitro transcription experiments using nuclei isolated from different mouse tissues. Having cloned the mouse and human CYP1A1 genes, these constructs will be included in the in vitro transcription assays as controls. To understand how this gene is regulated in vivo, gene constructs will be made and transgenic mice generated that carry the entire gene. These animals will be used in determining if the human CYP1A2 gene is inducible in a tissue specific and developmental fashion.